PROJECT SUMMARY Tumor-associated myeloid cells (TAMCs), which consist of tumor associated macrophages and myeloid- derived suppressor cells, make up a majority of cellular infiltrates in glioma. TAMCs are potently immunosuppressive, and represent a major barrier to successful immunotherapy. TAMCs highly express arginase-1 (Arg-1), a catabolic enzyme thought to deplete arginine from the tumor microenvironment. Despite being a well-known marker of immunosuppressive cells, the metabolic reasons for this choice are not clear. Examination of TAMCs phenotype in murine glioma models using RNA-seq, bulk metabolomics, and carbon-13 arginine flux revealed that two separate pathways of arginine catabolism converge on the generation of ornithine. Ornithine is the prerequisite substrate for the de-novo generation of polyamines, a group of nitrogen- rich metabolites with foundational importance to all domains of biology. Importantly, we found that the rate- limiting step of polyamine generation, ornithine decarboxylase 1 (ODC1), is dramatically upregulated by glioma infiltrating TAMCS, suggesting de-novo polyamine generation is important for their function. Therefore, the overall goal of this proposal is to determine how arginine is catabolized into polyamines by TAMCs, and to determine if inhibition of this metabolic pathway can enhance immunotherapy for glioma. Interestingly, we discovered that a second, unstudied metabolic pathway of arginine metabolism, the de- novo generation of creatine from ornithine, is preferentially utilized by TAMCs in our glioma models. The first aim of this project is to generate genetically engineered mice (GEM) to determine the role of the de-novo creatine metabolism in generating immunosuppression in mouse models of glioblastoma. This aim will dissect if creatine generation is used as a fuel to survive in brain tumors, or is being used to promote immunosuppression. Our preliminary experiments suggest that inhibition of the products of arginine metabolism, polyamines, can perturb TAMC immune suppression. This suggests that polyamines may be critical metabolites for the functions of TAMCs in brain tumors. Therefore, the second aim of this proposal is to probe the importance of polyamine metabolism by TAMCs in glioma. We will generate unique conditional knockout animal models to probe the importance of this pathway in the immunosuppressive functions of TAMCs in glioma. The third aim of this project is to determine if targeting of these pathways can be combined with standard of care for glioma to promote animal survival. First, we will test if a blood-brain-barrier permeable inhibitor of creatine kinase can be used to stymie TAMCs in glioma. Next, we will use well established inhibitors of polyamine generation and polyamine uptake in attempts to blunt TAMC mediated immunosuppression in glioma. Lastly, we will take the clinical inhibitors described above, and determine if they can be used to potentiate checkpoint immunotherapy for glioma.